Paired Peripheral Blood Samples Research Articles

Lung cancer clustering by identification of similarities and discrepancies of DNA copy numbers using maximal information coefficient.

Lung cancer is the second most diagnosed cancer and the first cause of cancer related death for men and women in the United States. Early detection is essential as patient survival is not optimal and recurrence rate is high. Copy number (CN) changes in cancer populations have been broadly investigated to identify CN gains and deletions associated with the cancer. In this research, the similarities between cancer and paired peripheral blood samples are identified using maximal information coefficient (MIC) and the spatial locations with substantially high MIC scores in each chromosome are used for clustering analysis. The results showed that a sizable reduction of feature set can be obtained using only a subset of locations with high MIC values. The clustering performance was evaluated using both true rate and normalized mutual information (NMI). Clustering results using the reduced feature set outperformed the performance of clustering using entire feature set in several chromosomes that are highly associated with lung cancer with several identified oncogenes.

Outcomes of human leukocyte antigen-matched related donor and haploidentical allogeneic hematopoietic cell transplantation recipients by immune profiles of recipients and donors

BackgroundHaploidentical (Haplo) allogeneic HCTs (alloHCT) have been used more frequently over the last decade as survival is similar to HLA-matched related donor (MRD) alloHCTs. ObjectiveWe aimed to identify donor and recipient immune signatures before alloHCT that are associated with clinically meaningful outcomes in MRD vs Haplo alloHCT recipients. Study DesignThis retrospective cohort study of 165 MRD (n=132) and Haplo (n=33) alloHCT recipients and their related donors between 2007-2019 with paired peripheral blood samples immunophenotyped for T-cell, B-cell, NK cell and dendritic cell (DC) subsets. Immune cells were quantified before alloHCT in donors and recipients; calculations of immune cell ratios were classified as high, intermediate, and low and analyzed with alloHCT outcomes. ResultsHaplo donors were younger than MRD donors (median: 35 vs 51 years), whereas Haplo recipients were older than MRD recipients (median: 68 vs 54 years), were more likely to have a Karnofsky Performance Score ≤70 (76% vs 57%), 3+ comorbidities (54% vs 47%), and were in complete remission prior to alloHCT (58% vs 42%).In MRD alloHCT, a lower ratio of CD4+ to CD8+ effector memory cells in the donor was associated with lower 4-yr overall survival (OS; 25% v 61%; p=0.009), lower 4-yr progression free survival (PFS; 25% v 58%; p=0.014) and higher incidence of 1-yr transplant-related mortality (TRM; 39% v 7%; p=0.009) in recipients. A higher ratio of CD8+ effector memory to total NK cells measured in MRD recipients was associated with a higher incidence of grade II-IV aGvHD (63% v 37%; p=0.004) but was not statistically significant for III-IV aGvHD (23% v 12%).In Haplo alloHCT, a lower ratio of total T-regulatory to CD4+ central memory cells in the donor was associated with lower 4-yr PFS (22% v 60%; p=0.0091). A higher ratio of CD4+ effector memory to CD8+ effector memory cells measured in Haplo recipients pre-alloHCT was associated with lower 4-yr OS (25% v 88%; p=0.0039). In both MRD and Haplo recipients, a higher ratio of CD4+ naïve to CD4+ central memory cells was associated with a higher incidence of grade II-IV aGvHD (64% v 38%; p=0.04). ConclusionEvaluation of pre-alloHCT immune signatures of the donor and recipient may influence clinically meaningful patient outcomes in both MRD and Haplo transplants.

Abstract 3710: Determining the efficacy of blood filtration in patients diagnosed with pancreatic cancer through the liquid biopsy

Abstract Cancer becomes lethal as it spreads from the primary site to the rest of the body. Circulating tumor cells (CTCs) are considered a marker of disease progression and have been associated with decreased overall survival. Blood filtration is one such method of removing cancer cells from circulation to improve patient prognosis. This study utilizes the liquid biopsy to assess the efficacy of a proprietary filtration device on the blood of patients with pancreatic ductal adenocarcinoma (PDAC) using the third generation high-definition single cell assay (HDSCA3.0) workflow. Blood samples were collected and analyzed to characterize the CTCs and other rare cells present before and after filtration. Examination of 6 paired portal (PoVB) samples demonstrated a statistically significant decrease in total rare cells, total CK+ cells, and epi.CTCs across all patients due to filtration of the blood. Furthermore, analysis of 2 paired peripheral blood (PB) samples showed a decrease in total rare cells, total CK+ cells, and specific phenotypes of rare cells upon filtration. These preliminary results demonstrate initial proof of concept that this filtration device can remove CTCs from the circulation, which may be used as a potential therapeutic device for PDAC patient care. Citation Format: Divya D. Suresh, George Courcoubetis, Emmeline Lin, Jeremy Mason, Nicholas Nissen, Srinivas Gaddam, Simon Lo, Stephen Pandol, Peter Kuhn, Stephanie Shishido. Determining the efficacy of blood filtration in patients diagnosed with pancreatic cancer through the liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3710.

Early prognosis prediction in acute myeloid and acute lymphoid leukemia patients using cell-free DNA concentration ratios.

Background: Cell-free DNA (cfDNA) is a promising biomarker for disease prediction in many cancers, including acute leukemia (acute myeloid leukemia [AML] and acute lymphoblastic leukemia [ALL]). This study investigated the role of cfDNA in predicting relapse or unfavorable outcomes in acute leukemia patients upon initial diagnosis. Methods: Paired peripheral blood samples of 25 patients with ALL and AML were compared at baseline and induction/follow-up and clinically correlated with clinicopathological and outcome variables according to the risk category. cfDNA was isolated using commercial cfDNA extraction kits. The probability of poor outcomes in high-risk groups and a cut-off value for risk stratification minimal residual disease (MRD) positivity and outcome prediction were derived. Results: Twenty-five patients diagnosed with AML and ALL were risk-stratified based on NCI risk stratification, and of these 25 patients, 4 patients were of standard risk (SR) and 1 patient was of intermediate risk (IR), while a majority of patients (80%) were of high risk (HR). Of these, four HR patients passed away. The ratio of cfDNA reduction at baseline and the end of induction was a strong predictor of poor outcomes in high-risk patients, regardless of the MRD status. A cfDNA ratio score of 2.6 or higher at diagnosis/remission predicted poor outcomes, with higher accuracy than conventional MRD detection by flow cytometry. Conclusion: A higher cfDNA ratio at diagnosis/remission or at baseline predicts poor outcomes in acute leukemia patients. This pilot study suggests that cfDNA ratio scoring may be a useful tool for predicting prognosis in acute leukemia patients, regardless of the MRD status.

Abstract PR002: Single-cell spatial analysis reveals microenvironmental features that contribute to immune discrepancies between adult and pediatric nasopharyngeal carcinomas

Abstract Background: Nasopharyngeal carcinoma (NPC) is an EBV-positive malignancy with high immune infiltrates but also high immunosuppressive activities. Pediatric NPC only constitutes 0.1-1% of the total NPC incidences and exhibits superior treatment responses and prognosis compared to adult NPC. Clinically, we consider that the differences in the tumor landscape between adult and pediatric NPC patients are the vital driver of such immune discrepancies. Therefore, we apply scRNA-seq and Visium spatial sequencing to primary pediatric NPC tissues and paired peripheral blood samples, and integrate our data with multiple independent adult NPC scRNA-seq cohorts. Furthermore, we establish a bioinformatics analysis pipeline that can calculate spatial cellular compositions and biological activities. Here, we demonstrate that low lipid metabolism, disrupted interactions between NPC cells and regulatory T cells (Tregs)/CD8+ T cells, and high infiltration of central stem memory T cells, contribute to more potent and enduring anti-tumor immunity in pediatric NPC patients, and these microenvironmental features might serve as new immunotherapeutic vulnerabilities. Method: 5' scRNA-seq coupled with TCR/BCR profiling was performed on 5 primary and treatment-naïve pediatric NPC tissues with 5 paired peripheral blood samples. Visium spatial-seq was performed on 4 paired pediatric and 7 adult NPC tissues. We incorporated our scRNA-seq cohort with three independent adult NPC cohorts. Our computational framework was integrated with existing analysis packages, including Seurat, Spacexr, and SpaCET, to analyze single-cell-referenced spatial data. The key results in our study were validated by multiplex IHC staining and flow cytometry on patient samples. Results: We established a multi-center NPC scRNA spatial cohort containing 503,021 cells from 69 samples, and 15,222 spatial spots from 11 samples. We developed a computational framework to calculate spatial co-localization and biological activities. We unveiled that lipid metabolism was elevated in the tumor-T cell core in adult NPC. Strong co-localizations between NPC cells and Tregs/CD8+ T cells were found in adult NPC, with spatially enriched CD70-CD27 and LGALS9-TIM3 interactions. This finding was corroborated by multiplex IHC staining of KRT19+ NPC cells, CD4+/FOXP3+ Tregs, and CD8+/PD-1/TIM-3+ exhausted T cells. We also identified and characterized novel IL7R+ central stem memory T cells in pediatric NPC tissues and blood, with strong resilience to immunosuppressive cues, including TGF-β, PEG2, and cyclic AMP, and could lead to enduring immunity and anti-PD-1/PD-L1 responses. Conclusion: We demonstrate that higher T-cell immunosuppression and exhaustion caused by aberrant lipid metabolism, tumor-T interactions, and a lower age-associated central stem memory T-cell pool, are vital drivers of immune discrepancies in adult and pediatric NPC patients. Thus, screening and targeting these molecular microenvironmental characteristics might help stratify immunotherapy responders and generate therapeutic benefits in NPC patients. Citation Format: Lanqi Gong, Yu Zhang, Grace Guan, Beibei Ru, Peng Jiang. Single-cell spatial analysis reveals microenvironmental features that contribute to immune discrepancies between adult and pediatric nasopharyngeal carcinomas [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR002.

Free from Drug Therapy in Multiple Myeloma (FREEDMM): MRD-Based Cessation of Maintenance

Background. Treatment of newly diagnosed multiple myeloma (NDMM) consists of induction therapy followed by autologous stem cell transplant (ASCT) and indefinite maintenance. Continuous lenalidomide carries both significant clinical and financial toxicity. Minimal residual disease (MRD) has proven to be a powerful surrogate for PFS and OS but is not yet used to guide treatment. We hypothesize that MRD could be used to predict a subset of patients who can safely stop maintenance therapy and enter into a disease surveillance phase. Study Design and Methods. This prospective phase 2 study (NCT 05192122) is designed to stop post-ASCT maintenance therapy once sustained MRD negativity is achieved. MM patients are eligible if they have completed ≥2 years of maintenance therapy post-ASCT, and meet IMWG criteria for ≥ PR. Bone marrow (BM) MRD testing is performed twice, one year apart, using NGS. Sustained MRD-negativity is defined at a threshold of 10 -6, with negative MRD sustained between the Year 1 and Year 2 interval. Patients with sustained MRD negativity and absence of new bony lesions on PET then undergo maintenance therapy cessation. MRD-positive patients are removed from study. In patients who discontinue maintenance, BM testing to re-assess for MRD is repeated yearly for 3 years. At the time of MRD testing, peripheral blood (PB) samples are collected for MALDI-TOF mass spectrometry testing and circulating multiple myeloma cell (CMMC) enumeration. These are done in parallel with MRD assessment. Health-related quality of life (HRQoL) using the EORTC QLQ-MY20 questionnaire is measured before and after maintenance cessation. This study plans to enroll 50 patients to meet the primary objective of assessing sustained MRD-negativity 1 year after maintenance cessation. For all patients, 5-mL PB and BM samples are collected, processed, and kept frozen until analysis. Cellular indexing of transcriptomes and epitopes (CITE-seq) to simultaneously quantify cell surface protein and transcriptome data is used to characterize differences in the immune microenvironment between MRD-negative, MRD-positive, as well as between patients with biochemical and/or clinical progression and patients with MRD-resurgence but biochemically and clinically stable. Preliminary Results. FREEDMM enrollment began December 2021, and 31 patients have been enrolled, 29 of whom have undergone Year 1 BM for MRD assessment. Median follow up of our cohort is 414 (0-577) days. The median age is 69.5 (40-78) years, and the majority are either Black (n=9; 29%) or Hispanic (n=11; 35%). 18 (58%) patients had R-ISS 1 or 2, while 2 patients had high risk disease with presence of 17p deletion. The median number of years of maintenance therapy completed prior to Year 1 BM is 3.4 (2-7.9) years. 28 (96.6%) were in CR at time of Year 1 BM. Year 1 MRD results are pending in 13 patients. At Year 1, 11 (68.8 %) of 16 patients were found to be MRD-negative, while 5 (31.3%) patients were found to be MRD-positive with a median of 206 (1-9,748) cells per 10 6 nucleated cells detected. 24 (85%) of 28 patients had a negative MALDI-TOF result at the time of the Year 1 BM. MALDI-TOF results were false negative in 3 MRD-positive patients. PB CMMC samples were concordant with MRD results in all 5 (100%) patients who were tested. There was concordance between MALDI-TOF and CMMC results in 9 (75%) of 12 patients. At year 2, 4 patients continued to be MRD negative, while 1 patient who was previously MRD-negative became MRD positive. At most recent follow-up, 3 (42.9%) of 7 MRD positive patients at Year 1 experienced clinical progression and were removed from the study. 2 (50%) of 4 patients with sustained MRD-negativity have undergone maintenance cessation, with the remaining 2 awaiting confirmation of MRD-negative results. 59 paired PB (n=29) and BM (n=30) samples have been collected for CITE-seq analysis. Pilot CITE-seq experiments captured 10,000-15,000 cells with 33,000-56,000 reads/ cell on one MRD-positive BM and paired PB sample. CITE-seq results for remaining patients will be presented at the conference. Conclusion: This phase 2 trial applies a novel clinical trial design with an adaptive strategy to explore use of MRD testing as a guide to maintenance therapy cessation in MM patients and represents one of the first studies introducing transcriptome analysis of the tumor and immune microenvironment in the maintenance setting. Ongoing results will be reported at the conference.

Comparing Phenotyping, Gene Expression, and Function of T Lymphocytes before Mobilization Chemotherapy Versus after Stem Cell Harvesting in Patients with B Cell Lymphoma and Multiple Myeloma

Chimeric Antigen Receptor T cell (CAR-T) therapy is an effective treatment for relapsed and refractory hematological diseases. However, a significant medical unmet need persists, particularly for patients at the relapse and refractory (R/R) status, who often experience leukopenia or lymphopenia during lymphocyte collection. This limitation poses considerable challenges to the successful implementation of CAR-T treatment, primarily due to the impaired function of T cells resulting from multiple lines of treatment and disease progression. As a consequence, the quality of the CAR-T cell product may be compromised. Before introducing CAR-T therapy, most patients with B cell lymphoma or multiple myeloma (MM) experienced mobilization chemotherapy and high-dose granulocyte colony-stimulating factor (G-CSF) administration for stem cell harvesting, followed by autologous stem cell transplantation as a salvage or consolidation treatment. Given that the procedure of lymphocyte collection for CAR T cell manufacturing is quite similar to stem cell harvesting, it becomes reasonable to explore the possibility of collecting lymphocytes immediately after the stem cell harvesting process. The advantage of this strategy is that patients are typically in a healthier condition with better T cell function compared to their status during R/R status. However, the effects of mobilization chemotherapy and high-dose G-CSF on T lymphocytes remain inadequately understood. To assess the feasibility of lymphocyte collection, it is essential to delve into the function of T lymphocytes at two critical time points: before mobilization chemotherapy compared to after stem cell harvesting. In this study, we recruited 19 patients with B cell lymphoma or MM. Paired peripheral blood samples were obtained before administering mobilization chemotherapy and after stem cell harvesting. Flow cytometry analysis using CCR7 and CD45RA markers was applied to classify T cell subsets, including T naive or T stem cell-like (TN/SCM, CCR7+/CD45RA+), T central memory (TCM, CCR7+/CD45RA-), T effector memory (TEM, CCR7-/C5RA-), and T effector (Teff, CCR7-/CD45RA+) (Figure A). The data indicated no significant difference in T cell phenotype between pre-mobilization chemotherapy and after stem cell harvesting. CD4and PD-1 expression were also analyzed and showed no significant difference between the two time points. To gain further insights, we performed RNA sequencing on 3 paired samples from patients with large B cell lymphoma (UPN#3, UPN#11, UPN#16). Through our observations, we noticed that these 3 paired samples could be clustered into 2 groups based on differential gene expression (Figure B), which corresponds to pre-mobilization chemotherapy versus post-stem cell harvesting status. Our findings strongly indicate that T cells at the pre-mobilization chemotherapy status exhibit significant differences in gene expression compared to those at the post-stem cell harvesting status. Specifically, T cells from post-stem cell harvesting show significantly higher expression of NIBANI, ALOX5AP, and GLUL compared to those from pre-mobilization. NIBANI plays a vital role in down-regulating apoptosis by interacting with the MDM2/p53/Bcl2/Bax pathway, promoting cell survival under stress-dependent conditions. GLUL is responsible for glutamine synthetase, and glutamine is an important source of biosynthetic precursors in active T cells. On the other hand, T cells from pre-mobilization show higher expression of TOX, DUSP4, and TGFBR3 compared to those from post-stem cell harvesting. TOX is a transcription factor involved in T cell exhaustion and senescence, while DUSP4 expression is associated with defective TCR signaling and accelerated T cell senescence. Our data indicate that the proportion of T naive or T stem cell-like phenotypes remains unchanged after mobilization chemotherapy and G-CSF injections during stem cell harvesting. RNA sequencing data further reveal that T cells at the post-stem cell harvesting status are associated with down-regulated apoptosis, favorable survival and activation, and reduced exhaustion and senescence compared to those at the pre-mobilization stage. This study suggests that instead of collecting lymphocytes during relapse or refractory status, collecting them right after stem cell harvesting may be a viable alternative.

Exploration of Multiomic Profiles and Biomarkers as Predictors of Neoadjuvant Chemoradiotherapy Responsiveness in Esophageal Squamous Cell Carcinoma

The current gold standard of care for resectable locally advanced esophageal cancer is neoadjuvant chemoradiotherapy (NCRT) followed by surgery. Given that only 30-40% of patients with esophageal squamous cell carcinoma (ESCC) achieved a pathologic complete response (pCR) following neoadjuvant chemoradiotherapy, it is critical to understand the biological basis of NCRT resistance in esophageal cancer and identify biomarkers for these patients in order to further personalize treatment plans. We aim to depict the biological landscape of ESCC responsiveness and resistance to neoadjuvant chemoradiotherapy. Endoscopic biopsied specimens of the primary tumors and paired peripheral blood samples were obtained from 24 patients before neoadjuvant chemoradiotherapy and tested for whole exosome sequencing, RNA sequencing, and DIA mass spectrometry. Genomic data were analyzed for significantly mutated genes, copy number alterations, MSI, TMB, and mutational signatures. Transcriptomics and proteomics data were used to examine differentially activated pathways. GSEA and ActivePathways were used for the single omics level and joint multi-omics analysis, respectively. Tumor microenvironment (TME) characteristics were deconvoluted by xCell upon RNA-seq data. Treatment resistance biomarkers were identified and validated in a separate cohort using mIHC. In the study cohort, 54% (13/24) of the patients achieved pCR. WES data suggested that FBXW7 was more frequently mutated in the pCR group (fisher test p-value = 0.029), and the most significant cytoband loss in the pCR group was 9p21.3 (qval = 0.001). Differences in TMB, MSI, and mutational signatures were not significant between groups. Combined transcriptomics and proteomics analysis revealed that type I interferon signaling pathways and RIG-I-like receptor signaling pathways(p<0.05) were enriched in non-pCR tumors. Esophageal cancer cohort RNA-seq data from TCGA verified the correlation between the genetic variances (FBXW7 mutation and 9p21.3 loss) and the decreased expression of type I interferon signaling pathway genes. In TME analysis, tolerogenic dendritic cells and exhausted T cell signatures were significantly enriched in non-pCR tumors, indicating an immunosuppressive status in treatment resistant patients. Based on proteomics PPI network and differential expression genes from RNA-seq data, a biomarker panel consisted of 12 proteins predictive of non-pCR tumors was identified: STAT1, EIF2AK2, MX1, BST2, TRIM21, SAMHD1, IFI44L, GBP1, PARP14, ISG15, IFIT3, and HLA-B. The expression of selected genes was validated by mIHC in an independent cohort. Through a multiomics approach, we described the biological characteristics of ESCC with distinct responses to neoadjuvant chemoradiotherapy and proposed a panel of 12 proteins as predictive biomarkers for non-pCR patients.

Cytotoxic T cell subsets in peripheral blood and cerebrospinal fluid from patients with multiple sclerosis

Using multicolor flow cytometry, the main cytotoxic T lymphocytes (Tcyt) subsets were identified, based on the expression of CD45RA and CD62L in paired samples of peripheral blood and cerebrospinal fluid from the patients during the relapse (n = 32) and remission (n = 20) of multiple sclerosis (MS), as well as in the peripheral blood samples of healthy volunteers (n = 51). During the relapse of MS, we have observed a decreased relative number of CD3+CD4+ cells and CD4/CD8 ratio in cerebrospinal liquor. In peripheral blood taken from the relapsed MS patients, we have found significant correlations between EDSS score and absolute counts (r = -0,430, p = 0.014), and with relative numbers of CD45RA+CD62L+Tcyt (r = -0,502, p = 0.003). In remission state of MS, the relative numbers of blood CD45RA-CD62L-Tcyt cells exhibited a significant decrease (p = 0.005) to 8.70% (6.51-11.63) against control group with 12.18% (10.38-15.24), although it did not significantly differ (p = 0.114) from the relapsed patients with 11.31% (8.28-13.90). Studies of liquor samples have shown that, during MS relapse, the percentage of CD45RA-CD62L-Tcyt was increased (p = 0.027) up to 8.16% (6.40-11.40), while in remission state these cells comprised only 6.49% (4.51-8.39) from the total CD3+ cell number. During relapse of MS, some positive correlations were revealed between the relative number of nave, CM, EM and TEMRA Tcyt from liquor, and the percentages, as well as contents of similar T cell subsets in peripheral blood samples. The inverse relationship between the level of EM Tcyt from liquor and peripheral blood naive cells showed the close relationship between these two Tcyt subsets and clinical manifestations of MS (i.e., scores of EDSS scale). During the remission period, most of these correlations are disrupted. Further investigations of cytotoxic T cells dynamics in peripheral blood and cerebrospinal fluid will help to approach the understanding of MS pathogenesis by revealing novel markers for the clinical prognosis in this disorder.

Clinical significance of determining the hypermethylation of the RUNX3 gene promoter and its cohypermethylation with the BRCA1 gene for patients with breast cancer.

The aim of this study was to assess the clinical significance of RUNX3 gene hypermethylation in the pathogenetic mechanisms of breast cancer in women, taking into account its cohypermethylation with the BRCA1 gene. This study included 74 women with newly diagnosed breast cancer (samples from female primary breast carcinomas and paired peripheral blood samples) and 62 women without oncological pathology-control group (peripheral blood samples). Epigenetic testing for hypermethylation status studying was performed in all samples on freshly collected material with the addition of a preservative before the storage and DNA isolation. Hypermethylation of the RUNX3 gene promoter region was detected in 71.6% samples of breast cancer tissue and in 35.13% samples of blood. The RUNX3 gene promoter region hypermethylation was significantly higher among breast cancer patients compared to the control group. The frequency of cohypermethylation in RUNX3 and BRCA1 genes was significantly increased in breast cancer tissues compared to the blood of patients. A significantly increased frequency of the hypermethylation of the RUNX3 gene promoter region and its cohypermethylation with the BRCA1 gene promoter region was found in tumor tissue and blood samples from patients with breast cancer, in contrast to the control group. The identified differences indicate the importance of further investigations of suppressor genes cohypermethylation in patients with breast cancer. Further large-scale studies are needed to find out whether the detected hypermethylation and cohypermethylation of the RUNX3 gene promoter region will have an impact on the treatment strategy in patients.

Single-cell clonal tracing of glandular and circulating T cells identifies a population of CD9+ CD8+ T cells in primary Sjogren's syndrome.

Primary Sjogren's syndrome (pSS) is a complex chronic autoimmune disease in which local tissue damage in exocrine glands is combined with broader systemic involvement across the body in tissues including the skin. These combined manifestations negatively impact patient health and quality of life. While studies have previously reported differences in immune cell composition in the peripheral blood of pSS patients relative to healthy control subjects, a detailed immune cell landscape of the damaged exocrine glands of these patients remains lacking. Through single-cell transcriptomics and repertoire sequencing of immune cells in paired peripheral blood samples and salivary gland biopsies, we present here a preliminary picture of adaptive immune response in pSS. We characterize a number of points of divergence between circulating and glandular immune responses that have been hitherto underappreciated, and identify a novel population of CD8+ CD9+ cells with tissue-residential properties that are highly enriched in the salivary glands of pSS patients. Through comparative analyses with other sequencing data, we also observe a potential connection between these cells and the tissue-resident memory cells found in cutaneous vasculitis lesions. Together, these results indicate a potential role for CD8+ CD9+ cells in mediating glandular and systemic effects associated with pSS and other autoimmune disorders.

Single cell characterisation of tissue homing CD4+ and CD8+ T cell clones in immune-mediated refractory arthritis

Abstract Background: Immune-mediated arthritis (IMA) is a group of autoimmune diseases where chronic inflammation primarily targets different joints in the body. Rheumatoid Arthritis (RA) is the most common subtype. In childhood, RA mostly manifests as juvenile idiopathic arthritis (JIA). In this study, we aimed to characterize the tissue homing inflammatory pathogenic T cells in patients with IMA. Methods: We collected paired synovial tissue (ST) and peripheral blood (PB) samples from three patients with IMA and analyzed CD45+, CD4+ and CD8+ sorted cells with paired single-cell RNA and TCRα/β sequencing and validated the findings with published datasets. Results: Despite being disparate diseases, CD4+ T cells in IMA represented uniform phenotype across patients with no major patient specific clusters. The ST cells were more cytotoxic (GZMB), highly activated (LAG3, PDCD1) and proliferative (NME1, FABP5, G0S2and MKI67). A modest clonal expansion within the Treg cluster was found in the ST compartment as compared to PB (p&amp;lt;0.05). Conversely, the CD8+ T cell clusters showed strong patient specific characteristics. All T cell clones enriched to the ST upregulated genes belonging to TNF receptor superfamilies, inflammation markers (LMNA, RGCC, CREM), and transcription factor NR4A2. Pathway enrichment analysis showed enrichment of the TNFA signalling via NFKB and apoptosis pathways. Conclusions: Our data suggests that in patients with IMA, the synovial tissue is dominated with cytotoxic, highly activated CD4+ T cells with proliferation-associated gene expression signature. Shared CD4+ and CD8+ T cell clones can be found between PB and ST, but they have a more proliferative and activated phenotype when trafficking from PB to ST. Supported by grants from University of Helsinki, Finland

T lymphocyte characteristics and immune repertoires in the epicardial adipose tissue of heart failure patients.

Epicardial adipose tissue (EAT) acts as an active immune organ and plays a critical role in the pathogenesis of heart failure (HF). However, the characteristics of immune cells in EAT of HF patients have rarely been elucidated. To identify key immune cells in EAT, an integrated bioinformatics analysis was performed on public datasets. EAT samples with paired subcutaneous adipose tissue (SAT), heart, and peripheral blood samples from HF patients were collected in validation experiments. T cell receptor (TCR) repertoire was assessed by high-throughput sequencing. The phenotypic characteristics and key effector molecules of T lymphocytes in EAT were assessed by flow cytometry and histological staining. Compared with SAT, EAT was enriched for immune activation-related genes and T lymphocytes. Compared with EAT from the controls, activation of T lymphocytes was more pronounced in EAT from HF patients. T lymphocytes in EAT of HF patients were enriched by highly expanded clonotypes and had greater TCR clonotype sharing with cardiac tissue relative to SAT. Experiments confirmed the abundance of IFN-γ+ effector memory T lymphocytes (TEM) in EAT of HF patients. CCL5 and GZMK were confirmed to be associated with T lymphocytes in EAT of HF patients. EAT of HF patients was characterized by pronounced immune activation of clonally expanded IFN-γ+ TEM and a generally higher degree of TCR clonotypes sharing with paired cardiac tissue.

Free from Maintenance Drug Therapy in Multiple Myeloma (The FREEDMM Trial): A Pilot Study of Minimal Residual Disease (MRD)-Driven Discontinuation of Maintenance

Background Induction therapy followed by autologous stem cell transplant (ASCT) and maintenance is the current standard of care for the treatment of newly diagnosed multiple myeloma (NDMM). However, indefinite maintenance therapy after ASCT carries both significant clinical and financial toxicities. While minimal residual disease (MRD) testing has become part of routine standard of care, its use in guiding treatment decisions, in particular cessation of therapy, has not been established in the maintenance setting. We hypothesize that MRD monitoring could be used to guide clinical decision making, justifying cessation of maintenance therapy in a sub-group of patients with sustained MRD-negative remission. Study Design and Methods This is a phase 2 single-arm adaptive design study (NCT 05192122) for decision-making regarding maintenance therapy (Figure 1). Patients are eligible if they have a diagnosis of active MM, have completed at least 2 years of maintenance therapy post-ASCT, and meet International Myeloma Working Group (IMWG) criteria for very good partial response (VGPR) or complete response (CR). MRD testing is performed twice, at least one year apart, on bone marrow aspirate using next-generation sequencing (NGS) and MRD-negative status is defined at a threshold of 10-6. Patients with negative PET scan in VGPR or CR and sustained (i.e., twice, 1 year apart) MRD negative status discontinue maintenance therapy. MRD-positive patients continue continue maintenance and are taken off study. In patients who are MRD-negative and discontinue maintenance therapy, MRD testing will be re-assessed yearly for 3 years. At each time of MRD testing, MALDI-TOF spectrometry and measurement of circulating multiple myeloma cells (CMMCs) will be done in parallel to compare against MRD results. Health-related quality of life (HRQoL) will be assessed throughout and results on maintenance compared with those after discontinuation using the EORTC QLQ-MY20 questionnaire. This study will enroll 50 patients to address the primary objective of assessment for sustained MRD-negative status at 12 months after stopping maintenance therapy. For all patients, a 5-mL peripheral blood and bone marrow sample is collected and stored at the time of each standard of care bone marrow biopsy and will be used to characterize the immune microenvironment by Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) of paired peripheral blood and bone marrow in MRD-negative and MRD-positive patients as well as of patients progressing after stopping therapy. Preliminary Results Enrollment into FREEDMM trial began in December 2021, and to date 20 patients in CR have undergone a first bone marrow for MRD assessment, with results pending in 9 patients. The median age is 66.5 (range: 39-76) years, and the majority of patients are either Black (n=7; 35%) or Hispanic (n=7; 35%). One patient has high risk disease (17p deletion). The median number of years of maintenance therapy completed is 4.2 (range: 2.14-7.9) years. 17 (85%) patients had a negative MALDI-TOF test at the time of the first marrow. Seven (63.6%) of 11 patients were found to be MRD-negative. MALDI-TOF failed to accurately correlate with MRD results in 2 patients. 40 paired peripheral blood (n=20) and bone marrow aspirate (n=20) samples have been collected for CITE-seq analysis. Pilot CITE-seq experiments captured 6,700-11,000 cells with between 38,000-68,000 reads per cell on one MRD-negative and one MRD-positive bone marrow aspirate and paired peripheral blood sample. CITE-seq results for these 20 patients will be presented at the conference. Conclusion: This phase 2 trial applies a novel clinical trial design with an adaptive strategy to explore use of MRD testing as a guide to discontinuing maintenance therapy in myeloma and represents one of the first studies to explore in parallel various measures of disease monitoring (i.e., MRD, MALDI-TOF, CMMCs, CITE-seq). Ongoing results will be reported at the conference. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Isolation and characterization of uterine leukocytes collected using a uterine swab technique.

Leukocytes from the maternal-fetal interface are a valuable tool to study local changes in immune function during pregnancy; however, sampling can be challenging due to inadequate tissue availability and the invasive nature of placental bed biopsy. Here, we aim to purify and characterize leukocytes from paired peripheral and uterine blood samples to assess whether a less invasive method of uterine blood collection could yield a population of enriched uterine leukocytes suitable for ex vivo and in vitro analyses. Human peripheral blood mononuclear cells (PBMC) and uterine blood mononuclear cells (UBMC) expressed from surgical gauze post C-section were isolated, and immunophenotypic information was acquired by multi-parameter flow cytometry. PBMC and UBMC were stained for markers used to define T and B lymphocytes, macrophages, regulatory T (TReg ) cells, and natural killer (NK) cells. Prime flow was performed to check expression and analysis of CD16- CD56++ and CD16- CD56++ NK transcripts in PBMC and UBMC samples. Immunophenotyping revealed that over 95% of both live PBMC and UBMC consisted of CD45+ leukocytes. Higher percentages of CD16- CD56++ , characterized as uterine NK (uNK) cells, were observed in UBMC samples as compared to PBMC samples (18.41% of CD45+ CD3- vs. 2.73%, respectively), suggesting that CD16- CD56++ cells were enriched in these samples. In UBMC, 49.64% of CD3-negative cells were of peripheral NK phenotype (CD16+ CD56++ ), suggesting infiltration of maternal peripheral NK (pNK) cell in the uterine interface. Intrauterine leukocytes, especially CD16- CD56++ NK cells, can be collected in sufficient numbers with increased purity by sampling the uterine cavity postdelivery with surgical gauze. Our results suggest that this non-invasive protocol is a useful sampling technique for isolating CD16- CD56++ cells, however, due to peripheral blood contamination, the NK cell yield could be lower compared to actual decidual or endometrial samples post-partum which is more invasive.

Abstract 1957: Dissecting CTC phenotypic heterogeneity for predictive biomarker identification and its association with clonal lineage through single-cell multi-omic profiling

Abstract Background: The mutation, selection, and adaptation of tumor cells along disease progression exhibits a spectrum of phenotypic and genotypic heterogeneity. The importance of distinguishing phenotypic states of CTC in addition to genomic alterations has been addressed for identifying predictive biomarkers and understanding CTC biology. Current liquid biopsy usually relies on only one phenotypic state of CTCs without further genomic validation of cancer cell identity. Here, we developed HDSCA3.0, a multi-omic platform, which could distinguish various phenotypic states of CTCs followed by genomic characterization. Methods: Paired peripheral blood (PB) and bone marrow aspirate (BMA) samples were collected prospectively from 80 metastatic castrate resistant prostate cancer (mCRPC) patients for retrospective analysis. Seventy-nine of them were part of Cabazitaxel With or Without Carboplatin Trial (NCT01505868) and one independent index patient was included with aggressive disease and unfavorable prognosis. CTCs were detected, classified, enumerated through a four-channel immunostaining assay (DAPI|Cytokeratin|Vimentin|CD45/CD31) and a computational pipeline followed by manual curation, and subjected to single-cell copy-number profiling for clonality analysis and aggressive variant prostate cancer molecular signature (AVPC-MS) detection i.e. 2+ defects in PTEN, RB1, and TP53 genes. Results: CTC subtypes were categorized from Cytokeratin-positive rare cell groups based on the presence of mesenchymal features and platelet attachment. Of 79 trial cases, 77 (97.5%) had CTCs, 24 (30.4%) were positive for platelet-coated CTCs (pc.CTCs) and 25 (38.5%) of 65 sequenced patients exhibited AVPC-MS in CTCs. Survival analysis indicated that the presence of pc.CTCs identified the subset of patients who were AVPC-MS-positive with the worst prognosis. In AVPC-MS-negative patients, its presence showed significant survival improvement from combination therapy. In index patient, we uniquely identified genetically clonal mesenchymal-like CTCs (mes.CTCs) and their presence was significantly associated with one subclone emerged along clonal lineage. Meanwhile, differences of CTC abundance and phenotypic diversity were observed between paired PB and BMA as well as genomic variations. Conclusion: Our findings suggest pc.CTCs and AVPC-MS in CTCs as a multi-omic predictive biomarker to stratify mCRPC subpopulations with the worst prognosis and the most significant benefit of additional platinum therapy and illustrate a robust approach to analyze intra-patient CTC genotypic and phenotypic heterogeneity and association. Citation Format: Shoujie Chai, Nicholas Matsumoto, Ryan Storgard, Chen-Ching Peng, Ana Aparicio, Benjamin Ormseth, Kate Rappard, Cunningham Kate, Anand Kolatkar, Rafael Nevarez, Kai Han Tu, Ching-Ju Hsu, Amin Naghdloo, Paymaneh Malihi, Liya Xu, Paul Corn, Amado Zurita-Saavedra, James Hicks, Carmen Ruiz-Velasco, Peter Kuhn. Dissecting CTC phenotypic heterogeneity for predictive biomarker identification and its association with clonal lineage through single-cell multi-omic profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1957.

Molecular features differences between patients with BRAF mutant melanoma and thyroid cancer.

e15128 Background: The efficacy of BRAF inhibitors varies extremely among patients with different cancers, such as malignant melanoma (MM) and differentiated thyroid carcinoma (TC) (constituting &gt; 95% of thyroid cancer). This study aims to investigate molecular biomarkers that may explain the efficacy difference of BRAF inhibitors in patients with MM and TC. Methods: Tumor tissue and paired peripheral blood samples from patients with MM and TC were sent for matched tumor-normal next-generation sequencing of 1021 cancer-related genes. MM and TC patients harboring known BRAF activating mutations were retrospectively recruited in this study. Concomitant mutations, tumor mutation burden (TMB) and other mutational features were compared between these two populations. Results: A total of 69 patients with MM and 315 patients with TC were included. The total and median number of mutations in these two groups were 503/6 and 605/1, respectively. Correspondingly, TMB was higher in MM compared with TC (median: 2.88 muts/Mb vs. 0.69 muts/Mb, p = 0.0002). Compared with TC, more copy number variants (CNV) (total number: 142 vs 5, p &lt; 0.001) and less single nucleotide variants (total number: 353 vs 569, p &lt; 0.001) were identified in MM. Most BRAF mutations in MM were located on V600, and other mutations including D594A/G/N, K483E/Q and K601E were identified. In TC, nearly all mutations were BRAF V600E. Excluding CNV and structural variants, the mutation frequency of 28 genes in MM was higher than TC. Pathway enrichment analysis suggested that 9 out of 28 genes (APC, ARID1B, ARID2, CDKN2A, CTNNB1, EGFR, PTEN, TERT, TP53) were significantly enriched on the hepatocellular carcinoma signaling. TERT, CDKN2A and PTEN were frequently mutated in MM, which may contribute to the efficacy difference of BRAF inhibitors in patients with MM and TC. Conclusions: This study found specific molecular features may explaining the efficacy difference of BRAF inhibitors in MM and TC, which requires further validation in patients treated with BRAF inhibitors.

Detection of MET amplification by droplet digital PCR in peripheral blood samples of non-small cell lung cancer.

Mesenchymal-epithelial transition (MET) amplification is one of the mechanisms accounting for the resistance of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in lung cancer patients, as well as the poor prognosis. Fluorescence in situ hybridization (FISH) is the most widely used method for MET amplification detection. However, it is inapplicable when tissue samples were unavailable. Herein, we assessed the value of droplet digital PCR (ddPCR) in MET copy number gain (CNG) detection in non-small cell lung cancer (NSCLC) patients treated with EGFR-TKIs. A total of 103 cancer tissues and the paired peripheral blood samples from NSCLC patients were collected for MET CNG detection using ddPCR. In parallel, MET amplification in tissue samples was verified by FISH. Also, the relationships between MET CNG and EGFR T790M, as well as the EGFR-TKI resistance were also evaluated using Chi-square or Fisher's exact tests. The concordance rate of ddPCR and FISH in detecting MET CNG in tissue samples was 100% (102/102), and it was 94.17% (97/103) for ddPCR method in detecting the MET CNG among peripheral blood and tissue samples. No statistical difference was observed between MET amplification and EGFR T790M (p = 0.65), while MET amplification rate was significantly increased in patients with resistance to third generations of EGFR-TKIs as compared with patients with resistance to first/second EGFR-TKIs (p < 0.05). ddPCR is an alternative method to detect MET CNG in both tissues and peripheral blood samples, which is of worthy in clinical promotion.

Mucosal-Associated Invariant T (MAIT) cells are highly activated in duodenal tissue of humans with Vibrio cholerae O1 infection: A preliminary report.

Mucosal-associated invariant T (MAIT) cells are unconventional T lymphocytes with a semi-conserved TCRα, activated by the presentation of vitamin B metabolites by the MHC-I related protein, MR1, and with diverse innate and adaptive effector functions. The role of MAIT cells in acute intestinal infections, especially at the mucosal level, is not well known. Here, we analyzed the presence and phenotype of MAIT cells in duodenal biopsies and paired peripheral blood samples, in patients during and after culture-confirmed Vibrio cholerae O1 infection. Immunohistochemical staining of duodenal biopsies from cholera patients (n = 5, median age 32 years, range 26-44, 1 female) identified MAIT cells in the lamina propria of the crypts, but not the villi. By flow cytometry (n = 10, median age 31 years, range 23-36, 1 female), we showed that duodenal MAIT cells are more activated than peripheral MAIT cells (p < 0.01 across time points), although there were no significant differences between duodenal MAIT cells at day 2 and day 30. We found fecal markers of intestinal permeability and inflammation to be correlated with the loss of duodenal (but not peripheral) MAIT cells, and single-cell sequencing revealed differing T cell receptor usage between the duodenal and peripheral blood MAIT cells. In this preliminary report limited by a small sample size, we show that MAIT cells are present in the lamina propria of the duodenum during V. cholerae infection, and more activated than those in the blood. Future work into the trafficking and tissue-resident function of MAIT cells is warranted.

Genomic and TCR profiling data reveal the distinct molecular traits in epithelial ovarian cancer histotypes.

Epithelial ovarian cancer (EOC) is classified into five major histotypes: high-grade serous (HGSOC), low-grade serous (LGSOC), clear cell (CCOC), endometrioid (ENOC), and mucinous (MOC). However, the landscape of molecular and immunological alterations in these histotypes, especially LGSOC, CCOC, ENOC, and MOC, is largely uncharacterized. We collected 101 treatment-naive EOC patients. The resected tumor tissues and paired preoperative peripheral blood samples were collected and subjected to target sequencing of 1021 cancer-associated genes and T cell repertoire sequencing. Distinct characteristics of mutations were identified among the five histotypes. Furthermore, tumor mutation burden (TMB) was found to be higher in CCOC and ENOC, but lower in LGSOC and HGSOC. Alterations associated with DNA damage repair (DDR) pathways and homologous recombination deficiencies (HRD) were prevalent in five histotypes. CCOC demonstrated increased level of T cell clonality compared with HSGOC. Interestingly, the proportion of the 100 most common T cell clones was associated with TMB and tumor neoantigen burden in CCOC, highlighting more sensitive anti-tumor responses in this histotype, which was also evidenced by the enhanced convergent recombination of T cell clones. These findings shed light on the molecular traits of genomic alteration and T cell repertoire in the five major EOC histotypes and may help optimize clinical management of EOC with different histotypes.